Polymerase Chain Reaction

Polymerase Chain Reaction or PCR is a laboratory procedure performed to ___ and ___ a ___ for genomic sequencing

detect, amplify, specific gene region

This process utilizes short DNA sequences or fragments called ___ complementary to the target region

primers

The amplification is initiated by a ___ wherein ___ to create new copies

Taq polymerase, primers and free nucleotides attach to the target region

target gene (___) will be amplified to serve as a ___ for species identification

Cytochrome C Oxidase Subunit 1/C01, molecular marker

The process of replicating the DNA in vitro was made possible by the discovery of the ___ by ___ and ___

structure of DNA, James Watson, Francis Crick

The process of PCR is a perfect example of ___

biomimicry

PCR, as a laboratory process, can be divided into two major parts:

1. preparation of the reaction cocktail

2. amplification procedure

The fine adjustment of these components is a process known as ___

optimization

T or F: PCR is identical to DNA replication

False, similar but not identical

DNA replication versus PCR

• DNA replication ⇒ replicates the entire genome (all the genetic composition of the material)

• PCR ⇒ amplifying only a portion of the DNA (particular sequence in our gene of interest) to produce thousands to millions of copies

3 specific stages of PCR

1. Denaturation

2. Annealing

3. Extension

8 reagents of the master/cocktail mix

1. DNA template

2. Primers

3. dNTPs (ATCG)

4. PCR Buffer (Tris, KCl, and MgCl₂)

5. Taq polymerase

6. MgCl₂

7. Ultrapure H₂O

8. Adjuvants

3 components of PCR Buffer

Tris, KCl, and MgCl₂

Formula for calculating cocktail mix

C₁V₁ = C₂V₂

What are the components in the formula for the cocktail mix

• C₁ ⇒ Stock concentration or initial concentration

• C₂ ⇒ Working concentration

• V₁ ⇒ Volume for 1 reaction

• V₂ ⇒ Total volume of reaction

Subunits of DNA structure

nucleotides

Nucleotide bases (4)

1. Adenine (A)

2. Thymine (T)

3. Guanine (G)

4. Cytosine (C)

Pairing of bases?

Complementary

Polarity or Directionality of DNA

3’ trunk end and 5’ tail end

Orientation of DNA

Antiparallel

Basic structure of DNA

Double-stranded helix

Specific stage where DNA replication occurs

S stage of interphase

Flow of information from DNA to RNA to proteins

Central dogma

Field of study: DNA → RNA

Transcriptomics

Field of study: RNA → protein

Proteomics

Field of study: DNA

Genomics

Field of study: Proteins → metabolites

Metabolomics

Key enzymes in DNA replication (7)

1. topoisomerase

2. helicase

3. single-strand binding proteins

4. DNA polymerase

5. primase

6. exonuclease

7. ligase

Differentiate topoisomerase I from topoisomerase II

• Type I topoisomerase cuts one strand of a DNA duplex, relaxation occurs, and then the cut strand is reannealed

• Type II topoisomerase cuts both strands of one DNA duplex, passes another unbroken DNA helix through it, and then reanneals the cut strands

Leading and lagging strand are collectively known as

Replication fork

Direction of DNA synthesis/assembling nucleotides in what direction?

5’ to 3’ end

Replication islets in lagging strand is known as

Okazaki fragments

Template strand is read in what direction

3’ to 5’

3 types of DNA polymerase in prokaryotes

1. DNA pol I

2. DNA pol II

3. DNA pol III

Function of DNA pol I

Exonuclease activity removes RNA primer and replaces with newly synthesized DNA

Function of DNA pol II

repair function

Function of DNA pol III

main enzyme that adds nucleotides in the 5’ to 3’ direction

Eukaryotic DNA polymerase for DNA replication/priming

alpha

Eukaryotic DNA polymerase for base excision repair (2)

beta and lambda

Eukaryotic DNA polymerase for mitochondrial DNA replication

gamma

Eukaryotic DNA polymerase for chromosomal replication with excision repair

delta

Eukaryotic DNA polymerase for chromosomal replication with repair

epsilon

Eukaryotic DNA polymerase for DNA repair

theta

Eukaryotic DNA polymerase for sister chromatid cohesion

sigma

Eukaryotic DNA polymerase for non-homologous end joining

mu

Transiently intertwined DNA molecules formed during DNA replication; two circular DNA molecules are interlocked

Catenanes

3 types of models of DNA replication

1. Semiconservative

2. Conservative

3. Dispersive

Who invented PCR

Kary B. Mullis

7 applications of PCR

1. Species Identification and Classification

2. Cloning

3. Forensic Biology

4. Population Genetics and Genetic Diversity

5. Genotyping

6. Gene Expression

7. Detection of Pathogens etc.

Name the 4 dNTPs

1. dATPs (deoxyadenosine triphosphate)

2. dCTPs (deoxycytidine triphosphate)

3. dGTPs (deoxyguanosine triphosphate)

4. dTTPs (deoxythymidine triphosphate)

What kick starts amplification

Primers

A pair of short DNA fragments that ___ with the DNA template and ___

hybridizes, defines the region that will be amplified

Why is CO1/COX1/MTCO1 gene used?

Highly conserved gene

Taq polymerase is derived for the thermophilic eubacteria

Thermus aquaticus

Habitat of Taq polymerase

hot sulfur springs

PCR in action, 6 steps

1. Initial denaturation

2. Amplification

   a. Denaturation

   b. Annealing

   c. Extension/elongation

3. Final extension/elongation

Temperature range and duration for initial denaturation

94˚-96˚C, 2 minutes

Temperature range and duration for denaturation

94˚-96˚C, 30 seconds

Range of cycles in PCR

25-40 cycles

Temperature and duration of annealing

PRIMER-SPECIFIC; 45˚C-60˚C (54˚C in Taq polymerase) for 1 minute

Temperature and duration of extension

72˚C for 1 minute

Temperature and duration of final extension

72˚C for 10 minutes

Temperature range for hold

4˚C—10˚C

Differentiate in-vivo from in-vitro DNA replication (5)

What is fidelity

Accuracy of polymerase to attach to the correct nucleotides

Faint bands beyond the DNA ladder in PCR

primer dimers

Cause of primer dimers (2)

1. Excess primers that did not anneal or attach

2. Some primers may also attach to each other if they are poorly designed

3 general guidelines for PCR

1. Aseptic Technique: Avoid Cross-contamination!

2. Double check your Master Mix Calculation

3. Avoid pipetting error!

Order in creating the cocktail mix

1. ddH₂O

2. PCR buffer

3. MgCl₂

4. dNTPs

5. Primers

6. Taq polymerase

Typical container for cocktail mix

1.5mL microcentrifuge tube

PCR products are known as

amplicons

How to check PCR results

agarose gel electrophoresis

In AGE, DNA fragments migrate toward?

Positive cathode, anode

How many base pairs can the primers amplify?

800 to 1000 base pairs for C01

Meaning of TBE

Tris-borate-EDTA

Concentration range of TBE and TAE

0.5x—1x

Meaning of TAE

Tris-acetate-EDTA

When to use TAE versus TBE?

1. TBE for separating larger fragments, cloning, and gel extraction

2. TAE for separating smaller fragments and longer AGE runs

4 loading dye options

1. Xylene cyanol

2. Bromophenol blue

3. Cresol red

4. Tartrazine

6 options for agarose concentration (w/v%)

1. 0.5%

2. 0.7%

3. 1.0%

4. 1.2%

5. 1.5%

6. 2.0%

DNA size range for 0.5%

1000 to 30,000 bp

DNA size range for 0.7%

800 to 12,000 bp

DNA size range for 1.0%

500 to 10,000 bp

DNA size range for 1.2%

400-7,000 bp

DNA size range for 1.5%

200-2,000 bp

DNA size range for 2.0%

50-2,000 bp

6 options for binding/fluorescent dyes

1. SYBR Gold

2. SYBR Green

3. Gel Red

4. SYBR Safe

5. EVA Green

High quality DNA AGE result?

• Dark DNA bands with no smearing

After DNA extraction, bands beyond the DNA ladder are?

RNA

After PCR, bands beyond the DNA ladder are?

Primer dimers

After PCR, bands that are not the target region

Non-specific Amplifications

Cause for Non-specific Amplifications

• Poorly-designed primers leads to a higher chance of amplifying a different gene

4 options for optimizing template DNA

1. Longer cycles

2. Fewer cycles

3. Dilution of DNA samples with contaminants brought about by the extraction method

4. Re-extract DNA

With longer PCR cycles, DNA template should be?

Diluted

With shorter PCR cycles, DNA template should be?

Increase concentration

Possible reasons for poor PCR attributed to template DNA (2)

1. Low quality DNA

2. Too much contaminants

Possible reasons for poor PCR attributed to primers (3)

1. Hairpin/dimer formation

2. Wrong melting temperature

3. Too low concentration

2 options for optimizing primers

1. Adjust primer concentration

2. Redesign primer

Ideal primer concentration range

0.05 µM to 1 µM

Possible reasons for poor PCR attributed to MgCl₂

Concentration is too low